Binding of l-[3H]glutamate to repeatedly frozen-thawed rat brain membranes |
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| Institution: | 1. Evolve BioSystems, Inc., Davis, CA 95618, USA;2. Department of Food Science and Technology, University of Nebraska, Lincoln, Lincoln, NE 68588-6205, USA;3. Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institutet, 17121 Solna, Sweden;4. Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 14152 Stockholm, Sweden;5. Department of Neonatology, Karolinska University Hospital, 14186 Stockholm, Sweden;6. Foods for Health Institute, University of California, Davis, Davis, CA 95616, USA;7. Department of Pediatrics, University of California Davis Children’s Hospital, Sacramento, CA 95817, USA;8. Department of Food Science and Technology, University of California, Davis, Davis, CA 95616, USA;9. Department of Nutrition, University of Nevada, Reno, Reno, NV 89557, USA;10. Pediatric Rheumatology, Karolinska University Hospital, 17176 Solna, Sweden;1. Key Laboratory of Conservation Tillage and Ecological Agriculture, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China;2. Key Laboratory of Terrestrial Ecosystem Carbon Neutrality, Liaoning Province, Shenyang 110016, China;3. University of Chinese Academy of Sciences, Beijing 100049, China;4. Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China;5. College of Agriculture, Ludong University, Yantai 264025, China;6. Shandong Agricultural University, Taian 271018, China;7. School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China;1. Key Laboratory of Advanced Drug Delivery Systems of Zhejiang Province, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China;2. Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China;3. Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712, USA;4. Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, TX 78249, USA;5. Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA;6. Zhejiang Laboratory of Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou 311121, China;7. MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China;1. Department of Medical Sciences, Clinical Diabetes and Metabolism, Uppsala University, Sweden;2. IIBm Alberto Sols (CSIC-UAM), Madrid, Spain;3. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Spain;1. GEMMA-Group of Environmental Engineering and Microbiology, Department of Civil and Environmental Engineering, Escola d′Enginyeria de Barcelona Est (EEBE), Universitat Politècnica de Catalunya-BarcelonaTech, Av. Eduard Maristany 16, Building C5.1, E-08019 Barcelona, Spain;2. GEMMA-Group of Environmental Engineering and Microbiology, Department of Civil and Environmental Engineering, Universitat Politècnica de Catalunya-BarcelonaTech, c/Jordi Girona 1-3, Building D1, E-08034 Barcelona, Spain |
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| Abstract: | l-3H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, KD = 697 nM; maximal binding capacity, Bmax = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (KD1 = 26 nM, Bmax1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (KD2 = 662 nM, Bmax2 = 10.5 pmol/mg protein). l-3H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors. |
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